Assay for the detection of proteases

ABSTRACT

The present invention relates to a method of detecting the presence of an active protease in a sample. The method involves the following steps: (1) providing a solid support to which is attached a ligand reactive with a protease; (2) contacting the solid support with a sample suspected to contain the protease; (3) washing the solid support to remove material not bound to the ligand; (4) contacting the washed solid support with a solution containing a peptide ester substrate which is hydrolysed for the protease, and (5) detecting protons produced by hydrolysis of the peptide ester substrate. In addition, the present invention provide a prostate specific antigen substrate, the substrate being of the general formula; A-B-Tyrosine-ester in which A is acetyl or carbobenzoxy and B is 2 to 6 amino acids.

ASSAY FOR THE DETECTION OF PROTEASE

The present invention relates to a an assay for the detection of activeproteases in samples, particularly samples of biological origin. Inaddition, the present invention relates to substrates having proteasespecificity.

During a study of mutant proteases the need arose for an assay methodthat could rapidly and accurately determine the activity and specificityof a large number of serine proteases produced in bacterial cultures.

Proteases of the serine (and thiol) families possess esterase activitieswhich proceed at much faster rates than the cleavage of peptide bonds.Advantage was taken of this activity to develop an "esterase" assaywhich uses a pH indicator to visualise the hydrogen ion release causedby the action of proteases on peptide-ester substrates. A series oftwenty compounds of the form, carbobenzoxy alanyl-X-methyl ester(Z-AX-OMe) were synthesised where "X" is one of the naturally occurringamino acids. These substrates were used in a 96 well microtitre plateassay to screen each mutant enzyme for activity and specificity. Thereactions were followed visually or quantified using a standard ELISAplate reader. The esterase assay, using trypsin as a test enzyme andZ-Ala-Arg-OMe as substrate, was more sensitive as than traditionaltrypsin substrates (three times the rate obtained with benzoyl arginineethyl aster and twenty times the rate of benzoyl argininep-nitroanilide) (Whittaker et al., Analytical Biochemistry, 220,238-243, 1994).

The success of this assay led us to question its possible broader use inthe characterisation and quantification of proteases in research ingeneral and in clinical enzymology in particular. For example the assaycould be useful for the rapid characterisation of a protease/s beingproduced by tumours where comparison of the reaction profile against aselected species of ester substrates would identify and quantify theproteases and potentially give an indication of the tumour's metastaticpotential.

A number of difficulties arise in using such an assay system forbiological samples. These include the buffering ions in the sample andthat some proteases are present at only very low levels. In order to atleast partly ameliorate theme problems and to improve the specificitythe present inventors have developed an assay method involving ligandcapture of the protease. In one form this is achieved by combining anELISA/Dot Immunobinding Assay (Allergy 50 (2); 119-125, 1995,Y. Yu) andthe Esterase Assay.

Accordingly, in a first aspect the present invention consists in amethod of detecting the presence of an active protease in a sample, themethod comprising the following steps:

(1) Providing a solid support to which is attached a ligand reactivewith a protease;

(2) Contacting the solid support with a sample suspected to contain theprotease;

(3) Washing the solid support to remove material not bound to theligand;

(4) contacting the washed solid support with a solution containing apeptide ester substrate which is hydrolysed by the protease; and

(5) Detecting protons produced by hydrolysis of the peptide estersubstrate.

The ligand may be any of a number of binding molecules well known in theart. It is however, necessary that the ligand binds to the protease at aposition which is not involved in the hydrolysis of the ester. It ispresently preferred that the ligand is an antibody, preferably amonoclonal antibody or antibody fragment.

The peptide ester substrate may be any of a number of substrateshydrolyzed by the protease. Preferably, the substrate is hydrolysedrapidly by the protease.

By appropriate selection of the peptide ester substrate it in possibleto add a further layer of specificity to the assay method. For example,use of ligand reactive with prostate specific antigen and a substratewhich is specifically hydrolysed by prostate specific antigen willresult in a highly specific assay method.

In a further preferred embodiment the production of a proton by thehydrolysis of the peptide ester is detected using a pH indicator. A pHindicator, such as phenol red, may be added after step 4, however, it ispresently preferred that indicator is included in the solution added instep 4.

In another preferred embodiment the sample is serum or semen.

The method of the present invention could be used to detect the presenceof a number of important mammalian proteases. These include:

    ______________________________________    1.           Tumour Markers                 Prostate specific antigen (PSA)                 Elastase - pancreatic antigen (PSA)                 Cathepsin B - breast cancer                 Urokinase type plasminogen activator    2.           COagulation                 Kallikrein                 Factors II, VII, IX, XI, XII                 Protein C                 Tissue plasminogen activator                 Urokinase                 Streptokinase                 Plasmin    3.           G I Tract Proteases                 Trypsin                 Chymotrypsin    4.           Complement factors    ______________________________________

In a preferred embodiment of the present invention the method is todetect the presence of prostate specific antigen in the sample.

As will be apparent to persons skilled in the art the method of thepresent invention is similar in some aspects to a conventional ELISA.There are, however, a number of important advantages over conventionalELISA techniques. The first is that the method of the present inventiondetects only active proteases. Secondly the method of the presentinvention does not require a second antibody stage.

The present inventors have also developed better substrates for prostatespecific antigen.

Accordingly in a second aspect, the present invention consists in aprostate specific antigen substrate, the substrate being of the generalformula:

    A-B-Tyrosine-ester

in which A is acetyl or carbobenzoxy and B is 2 to 6 amino acids.

In a preferred embodiment of the present invention A is acetyl and B isala-val.

In a further preferred embodiment the ester is a methyl aster.

In order that the present invention may be more clearly understoodpreferred forms thereof will now be described with reference to thefollowing examples and Figures in which:

FIG. 1 shows the specificity profile of PSA and the proteases present insemen. The similarity of the profiles indicates that PSA is a majorcomponent of semen with another enzyme(s) with arginine and lysinespecificity being present in the semen sample.

FIG. 2 shows semen and PSA profiles on substrates of the formAc-Ala-x-TyrOMe. The similarity of the profiles indicates that PSA ismost likely responsible for all the tyrosine activity in semen. PSA hada rate of 0.0263 OD₅₈₀ change/min/μg equivalent to 9.99 μmolessubstrate/min/mg. Semen was 0.0416OD₅₈₀ change/min/0.67 μl indicatingthat semen contained approx. 2.4 mg PSA/ml.

FIG. 3 shows activity of semen and chymotrypsin on substrates of theseries Ac-Ala-X-Tyr-OMe. The different profiles support the contentionthat all of the Tyr activity in semen is due to PSA.

FIG. 4 shows profiles of semen proteases on various substrates withvarying DMSO concentrations.

The present inventors produced a panel of substrates carbobenzoxyalanyl-X-methyl eater (Z-AX-OMe) where X is one of the naturallyoccurring amino acids. The reactivity of these substrates with a numberof serine proteases is set out in Table 1 and FIG. 1.

As expected from the literature these studies showed that of thesesubstrates the preferred substrate for prostate specific antigen (PSA)was Z-AY-OMe (FIG. 1).

The present inventors sought to develop a "better" substrate for PSA bysynthesising a range of substrates of the general formula:

    Acetyl-Ala-X-Tyr-methyl ester

In all eleven compounds were synthesised and purified and tested where Xwas the residue listed below. The rates obtained with semen and PSA onthese substrates are shown in FIG. 2.

                                      TABLE 1    __________________________________________________________________________    Esterase Rates of Selected Serine Proteases    Rates are A.sub.580 change/min./mg protease. Rates are also shown as a    percentage of the maximum activity to allow    comparison. For elastase and α-lytic protease the results are    normalised to alanine, the expected dominant preference.    Individual rates are approximately ± 10%.          Chymo-                          α-Lytic    P1    trypsin Trypsin Thrombin                                  Elastase                                          protease                                                  Subtilisin                                                          Proteinase K    Residue          Rate              %   Rate                      %   Rate                              %   Rate                                      %   Rate                                              %   Rate                                                      %   Rate                                                              %    __________________________________________________________________________    Ala   trace   0   0   0   0   15.7                                      100 6.2 100 1536                                                      73.8                                                          775 100    Cys   0.4 0.7 0   0   0   0   86.1                                      548.4                                          8.5 137.1                                                  1251                                                      58.2                                                          392 50.6    Asp   0   0   0   0   0   0   0   0   0   0   trace   18  2.3    Glu   0   0   0   0   0   0   0   0   0   0   83  3.9 97  12.5    Phe   37.8              68.5                  2.9 0.8 0   0   0   0   0   0   998 46.0                                                          342 44.1    Gly   0   0   0   0   0   0   0   0   0   0   22  1.0 58  7.5    His   1.5 2.7 3.0 0.8 trace   0   0   0   0   249 11.6                                                          203 26.2    Ile   0   0   0   0   0   0   1.7 11.0                                          0   0   16  0.7 5   0.6    Lys   1.5 2.7 155.8                      44.0                          4.3 49.0                                  0   0   0   0   153 7.1 61  7.9    Leu   2.4 4.3 0   0   0   0   3.4 21.7                                          0   0   1329                                                      61.8                                                          240 31.0    Met   6.6 12.0                  0   0   0   0   trace   2.0 32.3                                                  2149                                                      100 525 67.7    Asn   0.3 0.5 0   0   0   0   0   0   0   0   244 11.4                                                          137 17.7    Pro   0   0   0   0   0   0   0   0   0   0   trace   trace    Gln   0.5 0.9 0   0   0   0   0   0   0   0   1308                                                      60.9                                                          553 71.4    Arg   1.1 2.0 355.4                      100 8.7 100 0   0   0   0   97  4.5 24  3.1    Ser   trace   0   0   0   0   0.4 3.6 0.7 11.3                                                  366 17.0                                                          348 44.9    Thr   trace   0   0   0   0   3.1 19.7                                          4.2 67.7                                                  123 5.7 90  11.6    Val   0   0   0   0   0   0   2.1 13.4                                          1.3 21.0                                                  35  1.6 97  12.5    Trp   27.2              49.3                  trace   0   0   0   0   0   0   216 10.1                                                          353 45.5    Tyr   55.2              100 10.8                      3.0 0   0   0   0   0   0   345 16.1                                                          80  10.3    __________________________________________________________________________    SUBSTRATE                                X    __________________________________________________________________________    201                                      Ala (A)    204                                      Glu (E)    205                                      Phe (F)    208                                      Ile (I)    209                                      Lys (K)    213                                      Pro (P)    214                                      Gln (Q)    216                                      Ser (S)    218                                      Val (V)    219                                      Trp (W)    220                                      Tyr (Y)    __________________________________________________________________________

From FIG. 2, ten of the eleven substrates have improved activity overthe original screening substrate Z-AlaTyr-OMe) and two other simplesubstrates (Ac-Tyr-OMe and Ac-Ala-Tyr-OMe).

The substrate Ac-AlaValTyr-OMe (218) proved to be the best for PSA.(Another substrate Ac-AlaLeuTyr-OMe was made subsequently and it had 70%of the rate of substrate (218).

The rate with Ac-AlaValTyr-OMe, from the graph, was 15.5 faster thanZ-AlaTyr OMe (the screening substrate).

As shown in FIG. 2 the profile of PSA on the substrates was very similarto that obtained with semen indicating that the Tyr activity in san inpredominantly due to PSA. The profile with the sane substrate set iswidely diffrent from that obtained with chymotrypsin (FIG. 3) wherethere is no improvement in activity and widely varying rates on thevarious substrates. Clearly the more reactive substrates of thesubstrates are better reagents for an assay for PSA than was thescreening substrate Z-Ala-Tyr-OMe.

There are obviously further improvements that could be made to thesubstrates, i.e., the next logical step would be to vary P3 and then P4.We have made one longer substrate, Ac-Val AlaValTyr-OMe, which gave arate of 64.6% that of Ac-AlaValTyr-OMe. Obviously substituting Ac-Valfor Ac in the P3 position has a deleterious effect on the rate ofhydrolysis. This indicates that P3 and P4 are important and that othersubstitutions at P3 could possibly give a positive effective rateincrease.

In these studies, the present inventors also made the surprising findingthat activities of the serum proteases were optimal using substrates in25-30% DMSO as opposed to an expected optimum of 0% solvent (FIG. 4).

Combination of Antibody Capture of PSA with Subsequent Detection ofActive Protease via its Esterase Activity

Method:

Step a) Nitrocellulose dots were treated with a solution of monoclonalanti-PSA antibody (MP077, PJ301, 4C1 SCRIPPS) at 1 μL/dot and dried O/N.(These can be stored at =20° C. for many months).

Step b) The dots were blocked against non-specific protein capture by asolution of 0.5% casein in Tris buffered saline (TBS, pH7.4) for 2hours.

Step c) Diluted semen was applied to the dots and the PSA captured bythe. monoclonal antibody. Non-binding components were removed by washing(TBS×2, 50 mM Bicine, pH 8.5×2).

Step d) The dots were placed in microtitre plate wells and overlayedwith 40 μL Bicine buffer and 150 μL of 5 mM substrate 218 in 25% DMSOcontaining phenol red (concentrations as in normal assay).

Step e) The activity was detected visually by a colour change frompurple red to yellow. This could be quantified by transferring theliquids to an new plate and reading in a Microtitre plate Reader using afilter at 570 nM.

A further improvement of the method would be to allow the capture phaseand the esterase activity to be measured in the same vessel an in aconventional ELISA with the captured protease being detected bysubstrate overlay in the same well.

Experimental Design:

    ______________________________________                            No PSA        PSA                 Blank      capture No    capture           Blank nitrocellulose                            Ab.     semen + Semen           well  membrane   Control Control                                          Test           Dot 1 Dot 2      Dot 3   Dot 4 Dot 5    ______________________________________    Step a)  X       X          X     .check mark.                                            .check mark.    Capture    (αPSA)    Step b)  X       .check mark.                                .check mark.                                      .check mark.                                            .check mark.    Blocking    Step c) Semen             X       X          .check mark.                                      X     .check mark.    Step d)  .check mark.                     .check mark.                                .check mark.                                      .check mark.                                            .check mark.    Substrate    Results  -ve     -ve        -ve   -ve   +ve             Purple  Purple     Purple                                      Purple                                            Yellow                                            approx 2                                            days    ______________________________________

Conclusion

PSA was "captured" from semen using the nitrocellulose dot with anattached monoclonal antibody directed against PSA. Immobilised PSA is inan active form as measured by the ester assay result (Dot 5).

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

We claim:
 1. A method of detecting the presence of an active protease ina sample, the method comprising the following steps:(1) providing asolid support to which is attached a ligand reactive with a protease;(2) contacting the solid support with a sample suspected to contain theprotease; (3) washing the solid support to remove material not bound tothe ligand; (4) contacting the washed solid supported with a solutioncontaining a peptide ester substrate which is hydrolyzed by the proteasein which the peptide ester substrate is of the formula:

    A-B-Tyrosine-ester

in which A is acetyl or carbobenzoxy and B is 2 amino acids; and (5)detecting protons produced by hydrolysis of the peptide ester substrate.2. A method as claimed in claim 1 in which the ligand is an antibody orantibody fragment.
 3. A method as claimed in claim 2 in which theantibody is a monoclonal antibody.
 4. A method as claimed in claim 1 inwhich the production of a proton by the hydrolysis of the peptide esteris detected using a pH indicator.
 5. A method as claimed in claim 4 inwhich the solution added in step (4) further includes a pH indicator. 6.A method as claimed in claim 1 in which the sample is serum or semen. 7.A method as claimed in claim 6 in which the sample is semen.
 8. A methodas claimed in claim 1 in which A is acetyl and B is ala-val.
 9. A methodas claimed in claim 1 in which the ester is a methyl ester.
 10. Aprostate specific antigen substrate, the substrate being of the formula:

    A-B-Tyrosine-ester

in which A is acetyl or carbobenzoxy and B is 2 amino acids.
 11. Aprostate specific antigen substrate as claimed in claim 10 in which A isacetyl and B is ala-val.
 12. A prostate specific antigen substrate asclaimed in claim 10 in which the ester is a methyl ester.